Brain IGF-I regulates LTP, spatial memory, and sexual dimorphic behavior

A lack of brain-synthesized IGF-I almost completely abrogates hippocampal long-term potentiation (LTP), alters spatial memory and sex-dependent behavior, and produces major changes in the mouse hippocampal proteome.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** The manuscript presented by Herrero-Labrador et al shows how Igf-1 deletion significantly changes the hippocampal proteome on male and female mice and this conclusion was drawn after a significant reduction or oblation of LTP.
The work is sound and of relevance with minor and major issued to be solved: Minor issues: -there are small typos throughout the manuscript, please correct -in the text it is mentioned "p<0.05" while in some figures it is stated "p{less than or equal to}0.05". Please confirm in the entire manuscript which one was used and correct according. In some cases, there is repetition of this information in the text and in the figures captions. It is redundant. Please avoid it. -on page 6, " (Figure 2A,B)" is mentioned on the second sentence of the paragraph while the first sentence is already about figure 2. Please indicate each figure as soon as possible in the sentences.
- Figure 3 is quite short and I believe that it could have been merged with figure 2, making the necessary adjustments to the figure title and captions. -page 7 "No proteins were deregulated uniformly across both cohorts." Is repeated with the information inside the figure caption. -avoid "data not shown" as it cannot be peer-reviewed. Therefore, any sentence about missing data can not be evaluated. If needed, add this data as supplementary material.
-on the ethics section, please provide the authorization code for this project.
-explain the protein precipitation step in the sample preparation for proteomics -MilliQ water is a nonscientific term, and it refers to a brand. Authors are most likely referring to double deionized water (ddH2O).
-please follow the MIAPE guidelines with special care on indicating all software used in all steps (from data acquisition to data analysis) including their versions. P.e. Analyst is missing.
-The IHC section is indicated but there are no results based on this sample prep.
- Figure 1 Caption, please indicate that "*" refers to the analysis in relation to the base line while "#" refers to the analysis between the two experimental groups.
- Figure 2, is it "<" or "{less than or equal to}"? There is no text for two asterisks in the caption. Use just a straight line below the asterisk and center the beginning and end of the line with the center of the respective scatter data.
- Figure 4 doesn't have a title. For quicker analysis, please provide in supplementary material de same image but with the protein names aligned from both genders, to quickly observe that there are no differences in the trends of the same protein in both sexes. -Supplementary figure 1, please indicate the test which was used and retrieved no statistical differences.
Major issues: -could the authors clarify how the proteomics experiments were performed and why there are around 2000 proteins quantified in the female dataset and around 3300 proteins quantified in the male dataset? How was the normalization performed considering such a huge difference in the number of quantified proteins? - Figure 5 is quite complex to analyze and in some sense difficult to retrieve meaningful information. The authors could try to use other tools which allow a much quicker comparison of number of genes being regulated such as the one presented by Bonnot et al (DOI:10.21769/BioProtoc.3429) -validation of the results is very important and there are conclusions being drawn linking the observed phenotype and the proteomics changes. However, the electrophysiology experiments were performed in neurons and the proteome was performed in tissue, which comprises different cell types. It is important to understand if the proteins being modulated are in neurons or other cell type. In this case, IHC experiments on some of the most relevant proteins should be performed along with neuronal markers.
Reviewer #2 (Comments to the Authors (Required)): In their manuscript, Herrero-Labrador et al., investigate the effect of brain IGF-1 on neuronal activity, behavior and protein homeostasis. Herrero-Labrador and colleagues use the Nes-Cre mouse to delete IGF-1 in the brain and investigate long-term synaptic plasticity by recording excitatory postsynaptic field potentials from hippocampal slices. Here, brain slices of KO animals do not exhibit synaptic potentiation Further they investigate behavior and show that IGF-1 is an important factor for sex-specific behavior in the open field test. Lastly they analyze the proteome of hippocampal samples of control and KO animals. This analysis revealed that IGF-1 modulates the hippocampal proteome, which altered protein expression related to "vesicle-related compartments" and potentially also a link to sex hormone signaling.
While reading the manuscript, I recognized that all mouse experiments have been conducted at different ages. As age is an important confounder, it is therefore difficult to link alterations in neuronal activity (14-15 months) and protein homeostasis (10-15 months) to the observed differences in behavior (6-10 months).Unfortunately, the different parts of the manuscript are not well interconnected and rather seem as a line of different observations. While hippocampal slices are used to assess neuronal activity and protein homeostasis, the behavioral tests depend on motor function and exploration. Thus a classical memory test such as the Water maze test at the correct age should have been used to link molecular alterations in the hippocampus to the behavior.
I had the following issues with their presented data and conclusions in this manuscript.
1: Hippocampal-dependent behavior needs to be investigated at the same age, as neuronal activity and the proteome analysis has been performed.
2. The authors state that there is no differences between groups in the barnes maze test (Suppl. Fig.1). Yet only three female control mice have been investigated. This number is far too small to make hard conclusions. Overall the n is small for behavioral assessment (except male controls).
3: Important altered proteins ( Fig. 5-7) should have been confirmed by western blotting and tested whether they are regulated by sex hormones (in vitro). 4: Does brain IGF-1 differ between male and females in these animals? 4. Overall the figures are not precise enough described (especially Fig.5) making it very difficult to follow and understand reasons for the analysis. 5: In the methods part "tissue collection and immunohistochemistry" has been described, e.g. Hoechst and Prox1 staining, but no data have been shown. What is the reason for this?
Minor: There are several grammar mistakes in the manuscript.
At which specific dates have the behavioral tested been conducted?
The genetic background should be stated, as the authors can not ask all readers to read their first manuscript (Nieto-Estevez et al 2016b). Moreover the citations should be clearer defined as e.g. Nieto-Estevez et al 2016a and b has not been listed as 2016 a and b.

Point by point responses to the Reviewer's comments.
We would like to express our thanks to the Reviewers for their constructive comments and criticisms, which have allowed us to more clearly transmit the main message of our paper, i.e. that brain synthesized IGF-I is critical for hippocampal LTP, spatial memory formation and the regulation of sexual dimorphic behavior.
Furthermore, we show that brain IGF-I is necessary to maintain the structure of the granule cell layer, and that it regulates the inhibitory/excitatory balance and protein network modules that possibly have an impact on synaptic plasticity and memory.
Please, note that the major changes made in the manuscript are written in blue.

Reviewer #1
Thank you for your comments on our manuscript, please find our responses to all the issues you raised below.

Major issues:
(1) Could the authors clarify how the proteomics experiments were performed and why there are around 2000 proteins quantified in the female dataset and around 3300 proteins quantified in the male dataset? How was the normalization performed considering such a huge difference in the number of quantified proteins?
A unique normalization step encompassing all hippocampal samples was not possible mainly due to the different timing in the processing of both experimental groups. In that time, several changes/improvements to the massspectrometry phase were performed, obtaining more quantitative data in the female dataset, which led us to establish two independent processes of normalization. However, it is important to point out that male and female mice were age-matched when their brains were extracted. Thank you for pointing this out and to address this, we have first used a specific antibody to Prox1, a marker of granule neurons (which in the previous version was mentioned as data not shown). The results obtained indicate that brain Igf-I deletion produces significant increases in the number of ectopic Prox1 + neurons outside the granule cell layer (GCL) and specifically, in the molecular layer (ML) and the hilus (Hi) (Fig. 8). We suggest that this alteration may have a negative impact on the induction of LTP.
Next, we performed IHC experiments using a battery of antibodies against relevant proteins (RAB6A and gephyrin), proteins identified in the proteomic analysis, although we were unable to find a suitable antibody for IHC to study others (e.g., RAB2A). In addition, and as mentioned in the manuscript, interfacing the differentially expressed proteomes with the SYNGO and Ingenuity Pathway Analysis repositories revealed alterations to synaptic proteins and to proteins involved in LTP. Consequently, we also carried out IHC to detect presynaptic (synapsin I) and postsynaptic proteins (PSD95), as well as proteins with a relevant role in regulating the activity of inhibitory (GAD65 and PVA) and excitatory (VGLUT1) synaptic circuits. Moreover, since proteins involved in estrogen and androgen metabolism were functionally connected to some of the deregulated hippocampal proteins, we performed IHC to label aromatase (an enzyme catalyzing estrogen synthesis from androgens).
These results are now presented in the new Figures 10 and 11, and in the Supplementary Figures S6, S7, S8 and S9, and they lead us to draw two conclusions: 1) that brain Igf-I deletion could produce an imbalance in the inhibitory/excitatory ratio that affects LTP, and 2) that alterations to LTP and to synaptic protein modules may underlie the changes observed in synaptic plasticity and spatial memory.

Minor issues:
(1) There are small typos throughout the manuscript, please correct We apologize for this. The manuscript has now been carefully revised throughout.
(2) In the text it is mentioned "p<0.05" while in some figures it is stated "p{less than or equal to}0.05". Please confirm in the entire manuscript which one was used and correct according. In some cases, there is repetition of this information in the text and in the figures captions. It is redundant. Please avoid it.
We apologize for this inconsistency. We confirm that P<0.05, P<0.01, P<0.001 and P<0.0001 has been used in the statistical analysis.
(3) On page 6, " (Figure 2A,B)" is mentioned on the second sentence of the paragraph while the first sentence is already about figure 2. Please indicate each figure as soon as possible in the sentences.
This has now been modified accordingly. Thank you.
(4) Figure 3 is quite short and I believe that it could have been merged with figure 2, making the necessary adjustments to the figure title and captions.
The original figures 2 and 3 have now been merged into the new figure 4. Thank you for this suggestion.
(5) Page 7 "No proteins were deregulated uniformly across both cohorts." Is repeated with the information inside the figure caption.
I'm sorry, but we do not feel this idea is repeated in the figure caption.
(6) Avoid "data not shown" as it cannot be peer-reviewed. Therefore, any sentence about missing data cannot be evaluated. If needed, add this data as supplementary material.
As mentioned above, we have used a specific antibody to Prox1, a marker of granule neurons, data that was referred to as "not shown" in the previous version of the manuscript. The results indicate that brain Igf-I deletion produces a significant increase in the number of ectopic Prox1 + neurons located outside the granule cell layer (GCL), specifically, in the molecular layer (ML) and the hilus (Hi).
(7) On the ethics section, please provide the authorization code for this project.
The authorization code for this project is now provided in the first paragraph of the Materials and Methods.
(8) Explain the protein precipitation step in the sample preparation for proteomics According to the reviewer´s suggestion, the following sentence has been added to the Materials and Methods section: "Protein precipitation was performed using the ReadyPrep™ 2-D Cleanup Kit (Bio-Rad) following the manufacturer´s instructions".
(9) MilliQ water is a nonscientific term, and it refers to a brand. Authors are most likely referring to double deionized water (ddH2O).
We have now modified this accordingly. (12) Figure 1 Caption, please indicate that "*" refers to the analysis in relation to the base line while "#" refers to the analysis between the two experimental groups.
The meaning of the symbols has now been clarified in the legend to Figure 1. Thank you for pointing out this oversight.
(13) Figure 2, is it "<" or "{less than or equal to}"? There is no text for two asterisks in the caption. Use just a straight line below the asterisk and center the beginning and end of the line with the center of the respective scatter data.
We apologise for this inconsistency, it is *P<0.05 and **P<0.01, as now indicated in the legend to Figure 4.
The figure has now been modified as suggested by the reviewer.
(14) Figure 4 doesn't have a title. For quicker analysis, please provide in supplementary material de same image but with the protein names aligned from both genders, to quickly observe that there are no differences in the trends of the same protein in both sexes.
A title has been incorporated into Figure 4 (now Figure 5). Due to the experimental set-up, it was not possible to process hippocampal samples from male and female mice at the same time, although the animals were grouped by the age at which their brains were extracted (age-matched). During both independent analyses the mass spectrometer was cleaned, upgraded and several replacement parts were added, with the ensuing effect on performance. This is the main reason why it is not possible to produce a global heat-map representing all the proteomes quantified in male and female animals.
(15) Supplementary figure 1, please indicate the test which was used and retrieved no statistical differences.
The previous Supplementary figure 1 has been modified to include only the results obtained with the elevated plus maze (please see the response to Reviewer #2 below). Nevertheless, this data was analyzed using two-way ANOVA followed by a Bonferroni´s post hoc test.

Reviewer #2
Thank you for your constructive comments on our manuscript and please find our responses to all the issues you raised below.
(1) Hippocampal-dependent behavior needs to be investigated at the same age, as neuronal activity and the proteome analysis has been performed.
Following the reviewer´s comment, the performance of new mice was assessed in the Morris water maze (MWM) to address whether spatial learning and memory were altered by the lack of brain IGF-I. These mice were 6-16 months old, an age interval covering the ages of the mice used previously to assess neuronal activity (14-15 months), behavior (6-10 months) and protein homeostasis (10-15 months). The results obtained from the MWM assay indicated that Igf-I Δ/Δ mice had higher escape latencies, spent significantly less time and swam significantly shorter distances in the platform quadrant (P) than Igf-I Ctrl mice, further evidence that brain IGF-I plays a crucial role in the formation of new spatial memories. Moreover, our results indicate that the effect of brain IGF-I on spatial memory formation is partially sex-dependent. These new and important results are presented in Figures 2, 3 and Supplementary Figure S1, S2.
(2) The authors state that there is no differences between groups in the barnes maze test (Suppl. Fig.1). Yet only three female control mice have been investigated. This number is far too small to make hard conclusions. Overall the n is small for behavioral assessment (except male controls).
We agree with the Reviewer that using only three female control mice is too small a number to reach a clear conclusion. However, as indicated above we have now analyzed new animals (a total of 9 Igf-I Ctrl and 9 Igf-I Δ/Δ mice) using the MWM maze, which gave us more relevant results. As such, we have now removed the Barnes maze data from the manuscript.
(3) Important altered proteins (Fig. 5-7) should have been confirmed by western blotting and tested whether they are regulated by sex hormones (in vitro).
Thank you for pointing this out and as a result, we have now probed western blots with a battery of antibodies against relevant proteins identified in the proteomic analysis: RAB6A, RAB2A, Gephyrin, and VAMP7.
Although the relative protein levels showed a tendency to decrease in Igf-I Δ/Δ mice, these changes did not reach statistical significance (P<0.05, P value for RAB6A was 0.0554: Figure 9, Supplementary Figure S5). However, unfortunately we were unable to address whether these proteins are regulated by sex hormones in vitro.
Nevertheless, we did perform IHC experiments using antibodies against RAB6A and gephyrin, as mentioned above in response to Reviewer#1, yet for other proteins we could not find a suitable antibody for IHC (e.g.,

RAB2A
). In addition, and as mentioned in the manuscript, associating the differentially expressed proteomes with the SYNGO and Ingenuity Pathway Analysis repositories revealed alterations to synaptic proteins and proteins involved in LTP. Hence, we also carried out IHC to evaluate presynaptic (synapsin I) and postsynaptic proteins (PSD95), as well as proteins that play a relevant role in regulating the activity of inhibitory (GAD65 and PVA) and excitatory (VGLUT1) synaptic circuits. Moreover, since proteins involved in estrogen and androgen metabolism were functionally connected with some of the deregulated hippocampal proteins, we performed IHC to label aromatase (an enzyme catalyzing estrogen synthesis from androgens). These results are now presented in Figures 10 and 11, and in Supplementary Figures S6, S7, S8 and S9, and they lead us to conclude 1) that brain Igf-I deletion could produce an imbalance in the inhibitory/excitatory ratio that affects LTP, and 2) that alterations to LTP and to synaptic protein modules may underlie the changes observed in synaptic plasticity and spatial memory.
(4) Does brain IGF-1 differ between male and females in these animals?
We previously, analyzed the macroscopic phenotypes of the Nestin-Cre:Igf-I mice (Nieto-Estévez et al., 2016b) and we found that the Igf-I Δ/Δ mice had a similar body weight to the Igf-I Ctrl mice. We did not find differences in the volumes of any brain area studied, except that of the OB and the GCL of the DG, which were significantly smaller in the Igf-I Δ/Δ mice. Potential differences between male and female brains were not studied nor was the expression of IGF-1.
(5) Overall the figures are not precise enough described (especially Fig.5) making it very difficult to follow and understand reasons for the analysis.
We apologize for this. We believe that the data in the figures are now better described and the reasons for the analysis are more precisely defined in the new version of the manuscript. Following the comments of both reviewers, we decided to move the original Fig. 5 to the supplementary material (Fig. S4). The main message transmitted by this figure is that the alteration to membrane trafficking is common to males and females, and we consider that this is just as clearly presented in Fig. 4B.
(6) In the methods part "tissue collection and immunohistochemistry" has been described, e.g. Hoechst and Prox1 staining, but no data have been shown. What is the reason for this?
We apologise for this inconsistency. In the revised version of the manuscript we have used a specific antibody raised against Prox1, a marker of granule neurons (which in the previous version was mentioned as data not shown). The results indicate that brain Igf-I deletion produces a significant increase in the number of ectopic Prox1 + neurons located outside the granule cell layer (GCL), and specifically in the molecular layer (ML) and hilus (Hi). We suggest that this alteration could have a negative impact on LTP induction. Minor: (1) There are several grammar mistakes in the manuscript.
We apologize for this and the manuscript has now been carefully edited to correct any grammatical errors.
(2) At which specific dates have the behavioral tested been conducted?
The behavioral tests included in the first version of the manuscript were carried out between April 2017 and April 2018. The water maze test, and the data acquisition and analysis, was carried out from October 2022.
3. The genetic background should be stated, as the authors cannot ask all readers to read their first manuscript (Nieto-Estevez et al 2016b). Moreover, the citations should be clearer defined as e.g. Nieto-Estevez et al 2016a and b has not been listed as 2016 a and b.
The reviewer is correct and we now indicate that all the mice used in this study were on a C57Bl6N genetic background. Moreover, the citations are now clearer defined: Thank you for submitting your revised manuscript entitled "Brain IGF-I regulates LTP, spatial memory and sexual dimorphic behavior". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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